The following description provides a summary of information relevant to the present disclosure and is not a concession that any of the information provided or publications referenced herein is prior art to the claimed invention.
The SELEX process is a method for the in vitro selection of nucleic acid molecules that are able to bind with high specificity to target molecules and is described in U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands” and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Nucleic Acid Ligands” each of which is specifically incorporated by reference herein. These patents, collectively referred to herein as the SELEX Patents, describe methods for making an aptamer to any desired target molecule.
The basic SELEX process has been modified to achieve a number of specific objectives. For example, U.S. Pat. No. 5,707,796, entitled “Method for Selecting Nucleic Acids on the Basis of Structure” describes the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine” describes a method for identifying highly specific aptamers able to discriminate between closely related molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX” describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Pat. No. 5,496,938, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev” describes methods for obtaining improved aptamers after SELEX has been performed. U.S. Pat. No. 5,705,337, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX” describes methods for covalently linking an aptamer to its target. U.S. Pat. No. 6,376,424, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Tissue SELEX” describes methods to produce aptamers to cell or tissue specific markers without the purification of the specific marker.
The SELEX process encompasses the identification of high-affinity aptamers containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified aptamers containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5′- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, see supra, describes highly specific aptamers containing one or more nucleotides modified with 2′-amino (2′—NH2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe).
Further modifications of the SELEX process are described in U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,001,577, and U.S. Pat. No. 6,291,184, each of which is entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX”; see also, e.g., U.S. Pat. No. 6,458,539, entitled “Photoselection of Nucleic Acid Ligands”. These patents, collectively referred to herein as “the PhotoSELEX Patents” describe various SELEX methods for selecting aptamers containing photoreactive functional groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. The resulting photoreactive aptamers are referred to as photocrosslinking aptamers or photoaptamers.
Although these SELEX and photoSELEX processes are useful, there is always a need for processes that lead to improved properties of aptamers generated from in vitro selection techniques. For example, a need exists for aptamers to target molecules with better binding affinities than those achieved with naturally occurring DNA or RNA nucleotides, as well as methods for producing such aptamers. For many applications, such as for example, in vitro assays, diagnostics, therapeutic, or imaging applications, it is of interest to produce aptamers with slow dissociation rates from the aptamer/target affinity complex. Several techniques have been proposed for producing such reagents (see, e.g., WO 99/27133 and US 2005/0003362). However, these selection processes do not discriminate between the selection of reagents that have fast association kinetics with the target (i.e., fast on-rates) and the selection of reagents that have slow dissociation kinetics with the target (i.e., slow off-rates). Thus, there is a need for novel processes and techniques that favor the selection of slow off-rate aptamers while inhibiting the selection of aptamers that simply have a fast association rate with the target.
Finally, there is a need for aptamer constructs that include different built-in functionalities. These functionalities may include tags for immobilization, labels for detection, means to promote or control separation, etc.
Cytology consists of the evaluation of cell morphology, structure, and sub-structure, and as a diagnostic tool can be applied to any bodily fluid or organ. Specimens may be cells released in a fluid such as urine, gastric, sputum, pleural, spinal fluid, effusions, etc. or may be collected by needle biopsy or aspiration, scraping, or cytological brush. Aspiration biopsy may be performed on the lymph nodes, thyroid, salivary glands, breast, endometrial, or prostate. Cytological evaluations are used to evaluate organelle pathology, cell death (necrosis, apoptosis), cellular injury and response, cell aging, amyloidosis, autoimmune diseases, and to discriminate cancer from other disease states.
Histology consists of the evaluation of tissue morphology and structure for the diagnosis of a disease state, with the identification of malignancy being largely based on histological information. There are four major tissue categories in the body-epithelial, connective, muscle, and nervous. Direct microscopic visualization of tissue features is difficult due to the thickness of the tissue sample. Therefore techniques have been developed to allow the production of thin, representative sections of the tissue sample for subsequent analysis.
Both cytology and histology samples have been evaluated with immunological reagents. Antibodies to specific markers have been used to introduce dyes or other signaling moieties for visualization. Frequently, the immunological methods are more harsh than the standard methods because of the additional requirement to make the fixed sample permeable to the immunological reagents so the fixation method may be carefully balanced with the subsequent immunostaining to prevent the generation of artifacts. The size of the antibody limits diffusion into fixed cells and tissues. The Fc portion of the antibody may non-specifically associate with cell or tissue structures to generate erroneous results.
Cytologists and histologist are currently being asked to increase the number and range of tests conducted on a single collected specimen. To accomplish these goals it would be advantageous to have reagents that could provide one or more of the following characteristics: (1) be applied sequentially to the same sample without significant sample damage; (2) be pre-labeled to reduce or eliminate multiple process steps; (3) be pre-labeled with a number of different dyes or detectable moieties that can simultaneously be detected for detection of multiple targets from a single section; (4) eliminate the need for the antigen retrieval process as described below; (5) reduce or eliminate the permeabilization process; (6) reduce or eliminate non-specific association with the non-target; (7) and stabilize label location. Slow off-rate aptamers could address any of these needs in addition to providing (1) a more consistent and reliable reagent because they are chemically synthesized; (2) chemically robust reagents that have reduced storage requirements; and (3) rapid and high-throughput discovery of binding reagents to target new proteins.